**2. Patients and methods**

Between January 2005 and February 2008 a total of 100 patients were randomly selected from the lipid clinic and the carotid angioplasty clinic of a large tertiary hospital in Athens for inclusion in the study. Informed consent was obtained from each patient at recruitment according to our institutional policies. Eligible were patients with carotid artery stenosis from various causes and with a range of predisposing factors. Exclusion criteria included: acute cardiovascular disease, severe or unstable angina pectoris, clinically evident cardiac failure, severe arrhythmias, recent surgical procedures, inflammatory diseases, active liver disease or liver impairment, excessive alcohol consumption (>4 units/day) or history of alcohol abuse, known allergic reaction to statins, poorly controlled diabetes mellitus as defined by a haemo‐ globin A1c (HbA1c) level of >7mg/dl, uncontrolled hypertension indicated by systolic blood pressure (SBP) >140mmHg and/or diastolic pressure >85mmHg, history of deep vein throm‐ bosis, bleeding tendency, serum triglycerides >350mg/dl, evidence of thyroid dysfunction, use of systemic steroids or other anabolics, pernicious anaemia, impaired vitamin B12 or folate acid levels, abnormal serum urate at baseline, serum creatinine phosphokinase elevation of >1.5fold at baseline, pregnancy or lactation, and end-stage renal disease or dialysis.

range (reference range) in our lab was 31-61mU/l. Apolipoprotein A, B and Lp-a were measured using immuno-nephelometry with rabbit antisera (Dade Behring, Newark, USA) in

Oxidised Low Density Lipoprotein (LDL) Modification with Statin Therapy is Associated with...

http://dx.doi.org/10.5772/57188

131

The evaluation of stenosis was conducted by Triplex ultrasonography using an Apogee 800 plus scanner with a 7.5 MHz transducer (ATL Inc., Bothell WA, USA) at baseline and 12 months. The stenosis was calculated in three sections in each common and internal carotid artery, and the final measure was the mean value of the three. The value of stenosis in the most occluded vessel was used in the statistical analysis. Specifically, the internal carotid artery (ICA) and common carotid artery (CCA) bilaterally were evaluated for each patient using coloured and grey Doppler ultrasonography. An effort was made to completely visualize the vessels. Additionally, the pulse wave was estimated with Doppler phasmatometry as well as the blood flow velocity of the two vessels. Results were recorded in a validated form. Stenosis was defined as the presence of visual plaque in coloured or grey Doppler. The degree of stenosis was calculated by measuring the decrease of the lumen diameter and the maximum systolic blood flow velocity. In difficult cases, other parameters were taken into account, such as ICA/CCA max blood flow velocity ratio and the ICA end-diastolic velocity. A degree of stenosis >70% was considered as sever and angioplasty was advised. A degree of stenosis between 60 – 70% was defined as high, between 50 – 60% as moderate and <50% as mild. High, moderate and mild stenoses were treated conservatively. The intima media thickness (IMT) and plaque morphology were not studied due to specific lab requirements, not readily

Continuous variables were presented as mean values ± standard deviation, while qualitative variables were presented as absolute and relative frequencies. Normality tests were applied using the Kolmogorov-Smirnov criterion as well as Shapiro-Wilk test. Univariate analysis was initially applied to test the associations of oxLDL with carotid stenosis for each patient group as well as to identify first order correlations with various clinical parameters. Correlations between skewed continuous or discrete variables were evaluated using Spearman's pcoefficient, whereas correlations of normally distributed variables were evaluated by calcu‐ lating the Pearson's r-coefficient. Comparisons between normally distributed, continuous variables and categorical variables were made using the Student t-test. Analysis of categorical

The association of oxLDL with carotid stenosis was also tested through multiple Cox propor‐ tional hazard model. The results obtained were presented as Hazard Ratios (HR) and the 95% Confidence Intervals (CI). A backward elimination procedure was applied to all multivariate models (using P<5% as the threshold for removing a variable from the models). All models were adjusted for age, gender, SBP and TC. Kaplan-Meier curves concerning stenosis over the study period were plotted and Log rank test was performed. All reported P-values were based on two-sided tests and compared to a significance level of 5%. STATA 8.0 software (Stata

data was carried out with the [chi]2 test or Fischer's exact test when appropriate.

Corporation, 2003, Texas, USA) was used for the analysis.

a Dade Behring analyser.

available in our institution.

**2.1. Statistical analysis**

Patients were allocated into two groups according to the degree of carotid artery stenosis: those with arterial lumen occlusion of >70% in at least one common or internal carotid vessel consisted group A; those with stenosis <70% comprised group B. Patients in both groups were naive to statin therapy or if otherwise, a 6-month washout period was allowed before enrol‐ ment in the study. Group A underwent percutaneous transluminal carotid angioplasty with stenting by the same interventional cardiologist, prior to the initiation of statin therapy. Those patients were additionally administered clopidrogrel and salicylate. Both groups had to follow an American Heart Association step II diet and were encouraged to exercise.

All patients were placed on atorvastatin once daily at bedtime in individualised doses, tittered to achieve and maintain serum LDL cholesterol levels of <100mg/dl (and ideally <70mg/dl, if hypertension, renal impairment, smoking, hyperlipidemia, symptomatic peripheral arterial obstructive disease, or diabetes mellitus were present). The most common doses used to achieve the above levels of LDL ranged between 10 to 40mg, while seldom it was required to administer higher doses such as 60mg (median atorvastatin dose for the total population = 20mg, range 10 – 60mg). The use of other drugs known to act synergistically with statins causing rhabdomyolysis was prohibited during the study. Adverse events were assessed in every visit in a non-specific manner: every newly reported symptom was documented as possible adverse reaction due to statin therapy and subsequently evaluated by an expert in clinical biochemistry. Adherence to the medication regimen was assessed indirectly by the low LDL levels compared with baseline.

Medical anamnesis, anthropometrics, smoking habits, blood pressure, and laboratory inves‐ tigations comprising of complete blood count, fasting glucose, HbA1c, liver and kidney biochemistry, detailed lipid profile (TC, LDL cholesterol, high density lipoprotein [HDL] cholesterol, serum triglycerides [TG], apoB, and apolipoprotein A), urate, B12 and folate, thyroid function tests, homocysteine, Lp-a, and oxLDL were obtained at baseline and during follow-up visits, which were arranged at baseline, one, three, and six months; the final assessment was carried out in 12 months. Blood samples were collected after an at least 12 hour fast and a light, low-fat meal the night before sample collection was advised. Venous blood samples were collected in standard biochemistry vacutainer tubes. For the analysis of homocysteine and whole blood count, ethylenediaminetetraacetic acid (EDTA) vacutainer was used. Serum for biochemistry analysis was obtained by centrifugation (4000g) at 4°C for 7 min and was immediately tested.

Lipid profiles (TC, HDL, TG) were determined using commercially available enzymatic colourimetric methods (Dade Behring, Newark, USA) with a Dade Behring analyser. LDL was calculated with the use of Friedewald's formula as all had TG <350mg/dl [27]. For the meas‐ urement of circulating oxLDL, a commercially available kit (Mercodia, Uppsala, Sweden), based on a double antibody (4E6 and mouse monoclonal antiapoB) [28] capture ELISA test, was used. This method primarily detects malondialdehyde LDL (MDA-LDL). The normative range (reference range) in our lab was 31-61mU/l. Apolipoprotein A, B and Lp-a were measured using immuno-nephelometry with rabbit antisera (Dade Behring, Newark, USA) in a Dade Behring analyser.

The evaluation of stenosis was conducted by Triplex ultrasonography using an Apogee 800 plus scanner with a 7.5 MHz transducer (ATL Inc., Bothell WA, USA) at baseline and 12 months. The stenosis was calculated in three sections in each common and internal carotid artery, and the final measure was the mean value of the three. The value of stenosis in the most occluded vessel was used in the statistical analysis. Specifically, the internal carotid artery (ICA) and common carotid artery (CCA) bilaterally were evaluated for each patient using coloured and grey Doppler ultrasonography. An effort was made to completely visualize the vessels. Additionally, the pulse wave was estimated with Doppler phasmatometry as well as the blood flow velocity of the two vessels. Results were recorded in a validated form. Stenosis was defined as the presence of visual plaque in coloured or grey Doppler. The degree of stenosis was calculated by measuring the decrease of the lumen diameter and the maximum systolic blood flow velocity. In difficult cases, other parameters were taken into account, such as ICA/CCA max blood flow velocity ratio and the ICA end-diastolic velocity. A degree of stenosis >70% was considered as sever and angioplasty was advised. A degree of stenosis between 60 – 70% was defined as high, between 50 – 60% as moderate and <50% as mild. High, moderate and mild stenoses were treated conservatively. The intima media thickness (IMT) and plaque morphology were not studied due to specific lab requirements, not readily available in our institution.

#### **2.1. Statistical analysis**

bosis, bleeding tendency, serum triglycerides >350mg/dl, evidence of thyroid dysfunction, use of systemic steroids or other anabolics, pernicious anaemia, impaired vitamin B12 or folate acid levels, abnormal serum urate at baseline, serum creatinine phosphokinase elevation of

Patients were allocated into two groups according to the degree of carotid artery stenosis: those with arterial lumen occlusion of >70% in at least one common or internal carotid vessel consisted group A; those with stenosis <70% comprised group B. Patients in both groups were naive to statin therapy or if otherwise, a 6-month washout period was allowed before enrol‐ ment in the study. Group A underwent percutaneous transluminal carotid angioplasty with stenting by the same interventional cardiologist, prior to the initiation of statin therapy. Those patients were additionally administered clopidrogrel and salicylate. Both groups had to follow

All patients were placed on atorvastatin once daily at bedtime in individualised doses, tittered to achieve and maintain serum LDL cholesterol levels of <100mg/dl (and ideally <70mg/dl, if hypertension, renal impairment, smoking, hyperlipidemia, symptomatic peripheral arterial obstructive disease, or diabetes mellitus were present). The most common doses used to achieve the above levels of LDL ranged between 10 to 40mg, while seldom it was required to administer higher doses such as 60mg (median atorvastatin dose for the total population = 20mg, range 10 – 60mg). The use of other drugs known to act synergistically with statins causing rhabdomyolysis was prohibited during the study. Adverse events were assessed in every visit in a non-specific manner: every newly reported symptom was documented as possible adverse reaction due to statin therapy and subsequently evaluated by an expert in clinical biochemistry. Adherence to the medication regimen was assessed indirectly by the low

Medical anamnesis, anthropometrics, smoking habits, blood pressure, and laboratory inves‐ tigations comprising of complete blood count, fasting glucose, HbA1c, liver and kidney biochemistry, detailed lipid profile (TC, LDL cholesterol, high density lipoprotein [HDL] cholesterol, serum triglycerides [TG], apoB, and apolipoprotein A), urate, B12 and folate, thyroid function tests, homocysteine, Lp-a, and oxLDL were obtained at baseline and during follow-up visits, which were arranged at baseline, one, three, and six months; the final assessment was carried out in 12 months. Blood samples were collected after an at least 12 hour fast and a light, low-fat meal the night before sample collection was advised. Venous blood samples were collected in standard biochemistry vacutainer tubes. For the analysis of homocysteine and whole blood count, ethylenediaminetetraacetic acid (EDTA) vacutainer was used. Serum for biochemistry analysis was obtained by centrifugation (4000g) at 4°C for 7 min

Lipid profiles (TC, HDL, TG) were determined using commercially available enzymatic colourimetric methods (Dade Behring, Newark, USA) with a Dade Behring analyser. LDL was calculated with the use of Friedewald's formula as all had TG <350mg/dl [27]. For the meas‐ urement of circulating oxLDL, a commercially available kit (Mercodia, Uppsala, Sweden), based on a double antibody (4E6 and mouse monoclonal antiapoB) [28] capture ELISA test, was used. This method primarily detects malondialdehyde LDL (MDA-LDL). The normative

>1.5fold at baseline, pregnancy or lactation, and end-stage renal disease or dialysis.

an American Heart Association step II diet and were encouraged to exercise.

LDL levels compared with baseline.

130 Carotid Artery Disease - From Bench to Bedside and Beyond

and was immediately tested.

Continuous variables were presented as mean values ± standard deviation, while qualitative variables were presented as absolute and relative frequencies. Normality tests were applied using the Kolmogorov-Smirnov criterion as well as Shapiro-Wilk test. Univariate analysis was initially applied to test the associations of oxLDL with carotid stenosis for each patient group as well as to identify first order correlations with various clinical parameters. Correlations between skewed continuous or discrete variables were evaluated using Spearman's pcoefficient, whereas correlations of normally distributed variables were evaluated by calcu‐ lating the Pearson's r-coefficient. Comparisons between normally distributed, continuous variables and categorical variables were made using the Student t-test. Analysis of categorical data was carried out with the [chi]2 test or Fischer's exact test when appropriate.

The association of oxLDL with carotid stenosis was also tested through multiple Cox propor‐ tional hazard model. The results obtained were presented as Hazard Ratios (HR) and the 95% Confidence Intervals (CI). A backward elimination procedure was applied to all multivariate models (using P<5% as the threshold for removing a variable from the models). All models were adjusted for age, gender, SBP and TC. Kaplan-Meier curves concerning stenosis over the study period were plotted and Log rank test was performed. All reported P-values were based on two-sided tests and compared to a significance level of 5%. STATA 8.0 software (Stata Corporation, 2003, Texas, USA) was used for the analysis.
